Microbial communities play critical roles in fixing carbon from the atmosphere and fixing it in the soils. However, the large‐scale variations and drivers of these microbial communities remain poorly understood. Here, we conducted a large‐scale survey across China and found that soil autotrophic organisms are critical for explaining CO2 fluxes from the atmosphere to soils. In particular, we showed that large‐scale variations in CO2 fixation rates are highly correlated to those in autotrophic bacteria and phototrophic protists. Paddy soils, supporting a larger proportion of obligate bacterial and protist autotrophs, display four‐fold of CO2 fixation rates over upland and forest soils. Precipitation and pH, together with key ecological clusters of autotrophic microbes, also played important roles in controlling CO2 fixation. Our work provides a novel quantification on the contribution of terrestrial autotrophic microbes to soil CO2 fixation processes at a large scale, with implications for global carbon regulation under climate change. This study demonstrated that, on a large scale, soil autotrophic organisms are critical for explaining CO2 fluxes from the atmosphere to soils. Soil CO2 fixation rates are highly correlated to autotrophic bacteria and phototrophic protists. Paddy soils display four‐fold of CO2 fixation rates over upland and forest soils. Precipitation and pH, together with key ecological clusters of autotrophic microbes, also play important roles in controlling CO2 fixation.